Thermostable polymerases from Thermococcus pacificus

ABSTRACT

The invention relates to a thermostable polymerase based on  thermococcus pacificus ; DNA molecules which code for one such polymerase; expression vectors; host cells; methods for producing one such polymerase and the use thereof for polymerising nucleic acid, especially in the polymerase chain reaction.

This application is a United States national filing under 35 U.S.C. §371of international (PCT) application No. PCT/EP01/11529, filed Oct. 5,2001, designating the US, and claiming priority to German ApplicationNo. 100 49 211.8, filed Oct. 5, 2000.

The present invention relates to a thermostable polymerase fromThermococcus pacificus, DNA molecules which code for such a polymerase,expression vectors, host cells, process for preparing a polymerase ofthis kind and the use thereof for the polymerisation of nucleic acid,particularly in the polymerase chain reaction.

DNA polymerases are a family of enzymes which catalyse thepolymerisation of nucleic acids and play a part in both DNA replicationand in DNA repair. Thermostable DNA polymerases are frequently used inin-vitro processes, for example in the polymerase chain reaction (PCR),which has become an indispensable process in molecular biology. A commonproblem of the DNA polymerases used for this purpose is theincorporation of the wrong nucleotides during DNA synthesis, which leadsto mutated PCR products. This may cause problems in somemolecular-biological applications, particularly in the cloning andsubsequent recombinant expression of protein, as the mutationsintroduced may lead to inactive protein or protein which differs fromthe original protein in its properties. The mis-incorporation may becorrected by polymerases which have an inherent 3′-5′-exonucleaseactivity (so-called proofreading enzymes). The enzyme most commonly usedin PCR, Taq DNA polymerase, does not have this enzymatic activity, andit is known that the error rate of this enzyme is more than ten timeshigher than that of the proofreading enzymes (U.S. Pat. No. 5,545,552).

Various other known heat-stable DNA polymerases do indeed have aproofreading activity, but have other disadvantages (Lundberg et al.1991, Gene 108:1-6; EP 0 455 430; EP 0 701 000; WO 92/03556; WO92/09689). Thermostable DNA polymerases with 3′-5′-exonuclease activityare also known particularly from the organisms Thermococcus gorgonarius(WO 98/14590 A1) and the Archaeon strain KOD1 (EP 0 745 675 A2).

There is still a need for new thermostable DNA polymerases withproofreading activity and improved properties with regard to theirusefulness in molecular biology, particularly increased heat stability,3′-5′-exonuclease activity, and proofreading ability underPCR-conditions.

This problem is solved by the provision of a new thermostable DNApolymerase, obtainable from the organism Thermococcus pacificus.

The present invention relates particularly to a thermostable DNApolymerase from Thermococcus pacificus with 3′-5′-exonuclease activity.Preferably a DNA polymerase of this kind has the amino acid sequence SEQID NO: 2 (numerical code <210>2 or <400>2 in the attached sequencelisting).

In another aspect the present invention relates to a DNA molecule whichcodes for a thermostable DNA polymerase with 5′-3′-polymerase activity,and which

-   -   (a) contains the sequence SEQ ID NO: 1 (numerical code <210>1 or        <400>1 in the attached sequence listing) or the sequence        complementary thereto; or    -   (b) contains a sequence which constitutes a coherent fragment of        at least 60, preferably at least 90 nucleotides of SEQ ID NO:1        or the sequence complementary thereto; or    -   (c) contains a sequence which is so similar to SEQ ID NO: 1 or        the sequence complementary thereto that the DNA molecule        hybridises under stringent conditions with another DNA molecule        which contains SEQ ID NO: 1 or the sequence complementary        thereto; or    -   (d) contains a sequence which codes for a cohesive fragment of        at least 100 amino acids, preferably at least 200 amino acids of        the sequence SEQ ID NO: 2.

By stringent conditions are meant for the purposes of the presentinvention conditions under which two DNA molecules in a hybridisationexperiment hybridise with one another if they have a sequence identityof 97% or more, preferably 98% or more, most preferably 99% or more inthe hybridising section. The skilled man knows how to achieve suchconditions (Sambrook, Fritsch, Maniatis. Molecular Cloning. A LaboratoryManual, 1989. 9.47).

One way of obtaining a DNA molecule according to the invention is toisolate it from the organism Thermococcus pacificus. Thermococcuspacificus is known in the art (Miroshnichenko et al. 1998, Thermococcusgorgonarius sp. nov. and Thermococcus pacificus sp. nov.: heterotrophicextremely thermophilic archaea from New Zealand submarine hot vents.Int. J. Syst. Bacteriol. 48:23-29) and obtainable from publiccollections (DSMZ-Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH, Mascheroder Weg 1 b, 38124 Braunschweig, German, DSM No. 10394;American Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110, USA, ATCC 700653).

The organism may be cultivated in a manner known per se. A ready-mademedium produced by Messrs Becton Dickinson (Bacto Marine Broth 2216) maybe used. The medium may be made up in accordance with the manufacturer'sinstructions and the DSMZ (media 514 and 760); the addition of sulphurmay be omitted, with a slight loss of yield. The medium may be madeanaerobic by treatment with N₂ gas; this can be checked by addingresazurin (1 mg/l final concentration). In order to cultivate largervolumes of culture, for example, preliminary cultures may be used tobegin with. For these, 2 ml of the starting culture obtained from theDSMZ (DSM 10394) may be inoculated onto 20 ml of medium under anaerobicconditions. The incubation may take place overnight at 85° C. withoutshaking in culture flasks with an airtight seal. To start up the maincultures 20 ml of the preliminary culture may be anaerobicallyinoculated onto 500 ml of fresh medium and incubated under the sameconditions as the preliminary cultures.

The harvesting of the cultures may be done by transferring the culturesinto anaerobic centrifuge cups and then centrifuging the samples for 15min at 4° C. and 4000×g. The cell pellet thus obtained can then be usedfor the preparation of the genomic DNA by current methods.

The purified genomic DNA can be subject to a PCR reaction for examplewith the primers SEQ ID NO. 3, ATGATCCTCGATGCCGACTAC (Tpac3′) and SEQ IDNO. 4, TCATGTCTTAGGTTTTAGCCACGC (Tpac5′), in which the SEQ ID NO. 1 isamplified. The amplification product can then be purified by standardmethods, e.g. with the QIAquick PCR Purification Kit or QIAquick gelExtraction Kit (QIAGEN GmbH, Hilden, Germany).

Another method of producing a DNA molecule of the present inventionconsists in chemically synthesising such a DNA molecule. To do this, forexample, suitable oligonucleotides are first prepared by methods knownper se for synthesising oligonucleotides (e.g. Gait, M. J., 1984,Oligonucleotide Synthesis. A Practical Approach. IRL Press, Oxford, UK),from which a synthetic gene can be prepared by various methods. Suchmethods are known in the art (e.g. Stemmer et al. 1995, Single-stepassembly of a gene and entire plasmid from large numbers ofoligodeoxyribonucleotides, Gene 164(1): 49-53; Ye et al. 1992, Genesynthesis and expression in E. coli for pump, a human matrixmetalloproteinase, Biochem Biophys Res Commun 186(1):143-9; Hayden etMandecki 1988, Gene synthesis by serial cloning of oligonucleotides, DNA7(8): 571-7). In this way for example a DNA molecule with the sequenceSEQ ID NO: 1 may be prepared by methods known per se.

It is within the capabilities of the average man skilled in the art toprepare DNA molecules which are variants of a DNA molecule with the SEQID NO: 1 and similarly code for a thermostable DNA polymerase withessentially unaltered properties. Such variants differ from DNAmolecules of SEQ ID NO: 1 in that one, two, three, four, five, six,seven, eight, nine, ten or more nucleotides are deleted or substitutedby nucleotides with other bases, or additional nucleotides are insertedor added. By routine experiments the skilled man can determine whethersuch a modification has a significant influence on the properties of thepolypeptide coded by a DNA molecule of this kind. He has no difficultyfinding, by routine experiments, a large number of such variants whichcode for a thermostable DNA polymerase with essentially unalteredproperties. Such variants are therefore expressly included in theinvention.

Thus, the skilled man can in particular produce variants which aredegenerate compared with a DNA molecule with the sequence SEQ ID NO: 1in terms of the genetic code, i.e. DNA molecules with a nucleotidesequence other than SEQ ID NO: 1, but which code for the same amino acidsequence. This may be useful, when a DNA molecule of this kind isexpressed in a host cell, for optimising the codons with regard to thecodons of the host cell in question which are preferably used. Theskilled man will be aware of such processes.

It is also within the capabilities of the average person skilled in theart to delete smaller or larger sequence fragments, leading duringexpression to polypeptides with corresponding deletions in the aminoacid sequence. Using computer methods known per se based on comparing asequence, in this case SEQ ID NO: 1, with the sequences of other, wellcharacterised proteins, the skilled man can determine which sequencefragments (domains) are responsible for the enzymatic activities,polymerase or exonuclease activity. Outside these domains in particulardeletions (and of course substitutions as well) are possible, which haveno major effect on the enzymatic activity. It is easy to tell whether aparticular deletion in this sense has no effects.

It is also possible to modify the DNA molecule so that the DNApolymerase according to the invention takes the form of a fusion proteinwith additional amino acid sequences. Thus for example an affinitymarker in the form of a short peptide sequence may be attached to theC-terminus, for example a histidine hexamer which makes it easier topurify the protein (see below).

The skilled man may also deliberately switch off one of the twoenzymatic activities by mutation, for example by substitution ordeletion of one or more amino acids, or by deletion of an entiresequence fragment in the corresponding domain up to deletion of theentire domain. Thus, for example, the skilled man may prepare a DNAmolecule which codes for a polypeptide which has a polymerase, but notan exonuclease activity. This may be advantageous if a polypeptide ofthis kind is to be used for example in DNA sequencing.

In one particular embodiment, the DNA molecule according to theinvention codes for a polypeptide which has both 5′-3′-DNA polymeraseand 3′-5′-exonuclease activity.

In another aspect the present invention relates to a vector whichcontains a DNA molecule according to the invention, particularly anexpression vector. Preferably an expression vector of this kind has thefollowing features:

-   -   (a) one or more promoters;    -   (b) at least one operator which may be used to increase or        suppress gene expression;    -   (c) termination sequences for transcription and translation.

In an expression vector of this kind the DNA molecule according to theinvention is operatively connected with these features, with the resultthat the expression vector allows expression of the thermostable DNApolymerase according to the invention in a host cell. Expression vectorswhich are suitable for such purposes are known in large numbers in theprior art, as are methods of introducing the DNA molecule according tothe invention into such a vector, introducing the vector into hostcells, cultivating the host cells and isolating the polypeptide formed(cf. e.g. Sambrook et al., Molecular Cloning, 2^(nd) Ed., Cold SpringHarbour 1989, particularly Chapter 17). Suitable expression vectors arefor example the pQE vectors which may be obtained commercially fromMessrs Qiagen GmbH, 40724 Hilden, Germany. These vectors enable anaffinity marker, for example a histidine hexamer, to be incorporated atthe same time into the polypeptide formed, by means of which thepolypeptide can be purified easily and effectively. (Crowe et al., 1994,6xHis-Ni-NTA chromatography as a superior technique in recombinantprotein expression/purification, Methods Mol Biol. 31:371-87; Stüber etal. 1990, System for high-level production in Escherichia coli and rapidpurification of recombinant proteins, Immunol. Methods 4:121).

Accordingly, in another aspect, the present invention relates to a hostcell which contains such a vector. Preferably, the host cell isEscherichia coli.

In another aspect the present invention relates to a process forpreparing the DNA polymerase according to the invention, characterisedin that (a) host cells as described above are cultivated in a suitablemedium; and (b) the polypeptide formed is isolated from the medium orfrom the host cells.

In another aspect the present invention relates to a polypeptide whichcan be prepared by expression of a DNA molecule according to theinvention. In particular this is a polypeptide with a 5′-3′-DNApolymerase activity, preferably additionally with a 3′-5′-exonucleaseactivity.

The present invention also relates to a polypeptide which has a DNApolymerase activity, preferably a 5′-3′-DNA polymerase activity, andcontains a sequence which constitutes a cohesive fragment of at least100 amino acids, preferably at least 200 amino acids of the sequence SEQID NO: 2. A particularly preferred polypeptide is one with the sequenceSEQ ID NO: 2 or a polypeptide which contains the sequence SEQ ID NO: 2.

The thermostable DNA polymerase according to the invention mayadvantageously be used for the polymerisation of nucleic acid,particularly by polymerase chain reaction (PCR). It is highlythermostable, efficient and as a result of its proofreading activity hasonly a very small error content. Methods of polymerising nucleic acid bymatrix-dependent polymerisation of nucleotides using DNA polymerases ascatalysts, particularly polymerase chain reaction, are known in the art(cf. e.g. Sambrook et al., Molecular Cloning, 2^(nd) Ed., Cold SpringHarbour 1989, particularly Chapter 14).

In another aspect the present invention relates to a kit for use in thepolymerisation of nucleic acid, containing in separate containers

-   -   (a) a polypeptide of the present invention; and    -   (b) a reaction buffer for the polymerisation reaction.

Optionally a kit of this kind may additionally contain dATP, dGTP, dCTP,and dTTP, either as a mixture or in individual containers.

FIGURES

FIG. 1: Heat stability of Thermococcus pacificus DNA polymerase. Cf.Example 6. M: marker; −: empty trace; 0-90: preincubation of thepolymerase for 0, 5, 10 etc. minutes at 95° C.; A, B: M13 DNA singlestrand controls with no added polymerase.

FIG. 2: Detecting the 3′-5′ exonuclease activity of Thermococcuspacificus DNA polymerase. Cf. Example 7. Tpac: Thermococcus pacificuspolymerase; Pfu: Pyrococcus furiosus polymerase; exo⁻ Pfu: Pyrococcusfuriosus polymerase the 3′-5′ exonuclease of which is mutated so thatthere is no detectable exonuclease activity; +: addition of dNTPs; −:reaction mixture without dNTPs; 5, 10 . . . : incubation period inminutes.

FIG. 3: Detection of the proofreading ability of Thermococcus pacificusDNA polymerase under PCR conditions. Cf. Example 8. Tpac: Thermococcuspacificus polymerase; Pfu: Pyrococcus furiosus polymerase; exo⁻ Pfu:Pyrococcus furiosus polymerase, the 3′-5′ exonuclease of which ismutated so that there is no detectable exonuclease activity; A, B:characterise 2 independent PCR reactions; M: marker; +: digestion withBamHI takes place; −: undigested PCR fragment.

EXAMPLES Example 1 Culture of Thermococcus pacificus and Isolation ofGenomic DNA

The organism DSM No. 10394 was cultivated using a ready-made medium madeby Messrs Becton Dickinson (Bacto Marine Broth 2216). The medium wasprepared in accordance with the manufacturer's instructions andaccording to the DSMZ (Media 514 and 760) without the use of sulphur.The medium was made anaerobic by the use of N₂; this could be checked bythe addition of resazurin (1 mg/l final concentration).

First, preliminary cultures were prepared. For this, 2 ml of thestarting culture obtained from the DSMZ (DSM 10394) were inoculated onto20 ml medium under anaerobic conditions. The culture was incubatedovernight at 85° C. without agitation in culture flasks with an airtightseal. To start up the main cultures 20 ml of the preliminary culturewere anaerobically inoculated onto 500 ml of fresh medium and incubatedunder the same conditions as the preliminary cultures.

The cultures were harvested by transferring them into anaerobiccentrifuge cups and then centrifuging the samples for 15 min at 4° C.and 4000×g. The cell pellet thus obtained was then used for thepreparation of the genomic DNA using a commercially obtainablepurification kit (Qiagen Genomic-tip System, Qiagen GmbH, Hilden,Germany) in accordance with the manufacturer's instructions.

Example 2 Preparation of a Polymerase Expression Vector

The expression vector was prepared by generally knownmolecular-biological methods. The polymerase gene was isolated from thegenomic DNA of the organism Thermococcus pacificus by polymerase chainreaction (PCR). The primers used for this contained, in addition to thesequences homologous to the polymerase gene, a non-complementary nucleicacid sequence which coded for a restriction cutting site, so that byrestricting the amplified material and the expression vector theamplified material can be cloned into the expression vector.Oligonucleotides were obtained from Life Technologies GmbH, Karlsruhe,Germany. Other reagents for carrying out the polymerase chain reactionsuch as Taq DNA polymerase were obtained from QIAGEN GmbH, Hilden,Germany. The coding polymerase gene sequence SEQ ID NO: 1 was amplifiedusing 2.5 units Taq DNA polymerase or the proofreading DNA polymerasePfu DNA polymerase (Stratagene, Heidelberg, Germany). Also added to thereaction were oligonucleotides (0.2-1.0 μM) as primer, 200 μM of eachdNTP and 1× reaction buffer of the corresponding polymerase. A 3-stepPCR programme consisting of a denaturing step for melting the startingnucleic acid at about 94° C., a step of annealing of theoligonucleotides to their complementary DNA sequence at about 50-68° C.and an extension step at about 72° C. for amplification was carried out.Depending on the amount of starting nucleic acid, 30 to 40 amplificationcycles were carried out. After the PCR reaction the reaction product wasexamined on an agarose gel by comparison with a suitable DNA size markerto determine its specific length. PCR products of the expected size wereeither purified directly from the gel or from the PCR reaction.Commercially obtainable systems were used for this (QIAquick PCRPurification Kit or QIAquick gel Purification Kit, QIAGEN GmbH, Hilden,Germany). Purified PCR product and vector-DNA were cut with thecorresponding restriction enzymes and the reaction products were againpurified as described above. The vector-DNA used was the plasmid pQE80(QIAGEN GmbH, Hilden), which after the insertion of the target gene isable to express a fusion protein from a so-called His tag and the targetprotein. In the subsequent ligation reaction equimolar amounts ofvector-DNA and PCR product were used and ligated using T4-ligase (LifeTechnologies GmbH, Karlsruhe, Germany), the corresponding reactionbuffer and ATP overnight in a final volume of 20 μl at about 16° C.

Example 3 Preparation of a Bacterial Cell Expressing Thermococcuspacificus DNA Polymerase

1 to 2 μl of the ligation reaction were transformed intocalcium-competent DH5α-bacterial cells which optionally additionallycontained the plasmid pRep4 (QIAGEN GmbH, Hilden). Some of thetransformation reaction was then plated out on an agar plate whichcontained the antibiotic ampicillin and kanamycin or ampicillin on itsown as selection marker. The plates were incubated overnight for about15 to 18 hours at 37° C. Then colonies of bacteria were picked up usingsterile toothpicks or pipette tips, transferred into about 3 ml ofLB-medium with the appropriate antibiotic and incubated overnight at 37°C. Plasmids were isolated the next day according to the manufacturer'sinstructions with commercially obtainable kits such as the QIAprep MiniKit or the QIAGEN Plasmid Tips (QIAGEN GmbH, Hilden, Germany). Plasmidswere then checked using suitable restriction enzymes and sequencing tosee whether they contained the polymerase gene.

Example 4 Expression and Purification of the DNA Polymerase ofThermococcus pacificus

A construct which contained the error-free nucleic acid sequence of theDNA polymerase was transformed into DH5α/pRep4 competent cells. Cellswere cultivated in the presence of ampicillin and kanamycin in NZ-aminemedium and the expression of the polymerase gene was induced by theaddition of IPTG. After the bacterial cells had been harvested, theywere lysed using lysozyme, ultrasound and brief decoction. Thepolymerase protein with a His tag was selectively purified using acommercially obtainable purification kit (QIAexpress proteinPurification System, QIAGEN GmbH, Hilden, Germany) according to themanufacturer's instructions by metal affinity chromatography withnickel-NTA-agarose. The protein eluted with imidazole was dialysedagainst a storage buffer which consisted of 20 mM TrisHCl (pH 8 at 20°C.), 100 mM KCl, 1 mM EDTA, 0.5% (v/v) Nonidet P-40 substitute, 0.5%(v/v) Tween 20 and 50% (v/v) glycerol. The polymerase was stored in thisbuffer at −20° C.

Example 5 5′-3′ Polymerase Activity of Thermococcus pacificus DNAPolymerase

To demonstrate that the cloned nucleic acid sequence codes for a DNApolymerase, a test was carried out to check for DNA polymerase activity.The assay shows the extension of an oligonucleotide which is hybridisedto single-stranded M13-DNA. If the primer is extended, which can only bedone if the protein preparation added has a DNA polymerase activity, adouble-stranded DNA molecule is formed from the single-stranded startingnucleic acid. The activity is then detected on an agarose gel by meansof a difference in migration of the double-stranded DNA compared withthe single-stranded starting DNA. The extension rate is dependent on thepolymerase used. The quantity of end product of double-stranded DNA isdependent on the amount of DNA polymerase, the polymerase-specificextension rate and the time taken to carry out the reaction.

All the polymerisation reactions contained 50 ng of M13 mp18-DNA (20fmol; 7250 bases), 0.1 μM 30-mer oligonucleotide of the sequence5′-TTTCCCAGTCACGACGTTGTAAAACGACGG-3′ (SEQ ID NO: 5) and 50 μM of eachdNTP in 10 μl of 10 mM TrisHCl. Polymerisation reactions containeddifferent amounts of Taq DNA polymerase (0.2, 0.1, 0.05 and 0.01 units;QIAGEN GmbH, Hilden, Germany) or the polymerase preparation to be testedin various dilutions. The reaction was carried out for both enzymes in1× reaction buffer of Taq DNA polymerase (QIAGEN GmbH, Hilden), to which1 μg/ml BSA was added, in order to saturate any non-specific proteinbinding sites on the surface of the reaction vessel.

The polymerisation reaction was carried out in a model PTC-200Thermocycler made by MJ Research (Biozym, Hess. Oldendorf, Germany). Thereaction conditions were chosen as follows: 1 sec denaturing to dissolveany secondary structures present in the DNA, 30 sec hybridisation of theoligonucleotide at 55° C., followed by the primer extension at 72° C.for 3 min.

After the reactions had ended the reaction products were mixed with 1 μlof gel loading buffer (50% glycerol, 1× TAE buffer, 0.02 mg/mlbromophenol blue) and applied to a 1% agarose gel which contained 0.5μg/ml ethidium bromide to stain the DNA. The gel was analysed at 80 mAfor about 15 min in 1× TAE buffer, to separate the single-stranded anddouble-stranded DNA. The results show that the protein preparationobtained after the expression of the SEQ ID NO:1 has a DNA-dependent DNApolymerase activity.

Example 6 Heat Stability of Thermococcus pacificus DNA Polymerase

The heat stability of the polymerase was examined using the primerextension test described in Example 6. For this purpose, as amodification of the assay described above, 1 unit of the polymerasepreparation was preincubated for different lengths of time at 95° C. (0min, 5 min, 10 min, 15 min, 30 min, 60 min and 90 min). Then the assaywas carried out as described in Example 5 with these differentlypretreated polymerase preparations. In a modification of the assaysdescribed in Example 5 the buffer of Pfu DNA polymerase (Stratagene,Heidelberg) was used as the 1× reaction buffer. The results are shown inFIG. 1. Two control reactions which contained no DNA polymerase werealso carried out. The results clearly show that the polymerase showsabsolutely no loss of activity even after 90 min incubation, i.e. it hasextremely high heat stability (as a comparison: Taq DNA polymerase losesabout 50% of the polymerase activity after 60 min incubation at 94° C.).

Example 7 Detection of the 3′-5′ Exonuclease Activity of Thermococcuspacificus DNA Polymerase

The test described as follows was intended to examine to what extent theThermococcus pacificus DNA polymerase has a 3′-5′ exonuclease activity.The error rate with which a DNA polymerase duplicates the parent DNAduring the replication of the chromosomal DNA is dependent on a numberof factors: on the one hand, the choice of base complementary to thestarting nucleic acid is crucial, as well as the extent to which awrongly incorporated nucleotide can have another nucleotide of thepolymerase attached to it, and how much 3′-5′ exonuclease activity thepolymerase has. Using this exonuclease activity, wrongly incorporatednucleotides can be hydrolytically cleaved, so that a nucleotideincorporated by mistake can be replaced by the correct complementarynucleotide. This enzyme activity is also known as the proofreadingability of a polymerase.

The 3′-5′ exonuclease activity is detected by means of the hydrolyticbreakdown of a DNA. For this purpose the following substances were mixedtogether: 1 μg DNA size marker VI (Roche Biochemicals), optionally 200μM of each dNTP, 1× Pfu DNA polymerase reaction buffer (Stratagene,Heidelberg) and 1 unit of Thermococcus pacificus DNA polymerase.Reactions were set up with and without nucleotides: in the absence ofnucleotides the exonuclease activity is predominant, whereas it isinhibited by the addition of dNTPs. As a positive control, 1 unit of PfuDNA polymerase was used (Stratagene, Heidelberg), having a 3′-5′exonuclease activity, while the negative control used was Pfu Exo minusDNA polymerase (Stratagene, Heidelberg), the exonuclease activity ofwhich is sharply reduced by point mutagenesis. The reactions wereincubated for different periods (5 min, 10 min, 30 min, 60 min and 90min) at 72° C. and then analysed on a 1% agarose gel. The results shownin FIG. 2 clearly demonstrate that the Thermococcus pacificus DNApolymerase has a 3′-5′ exonuclease activity many times greater than thatof Pfu DNA polymerase.

Example 8 Detecting the Proofreading Ability of Thermococcus pacificusDNA Polymerase Under PCR Conditions

Extremely thermostable DNA polymerases with proofreading ability areused in particular in PCR reactions which are intended for thepreparation of mutation-free DNA fragments. This is particularlyimportant for certain molecular-biological applications such as thecloning of error-free PCR fragments for protein expression. DNApolymerases which have a 3′-5′ exonuclease activity synthesise PCRproducts with an error rate up to 12 times lower than Taq DNApolymerase, the standard enzyme for PCR applications.

In the present test the ability of Thermococcus pacificus DNA polymeraseto correct errors under PCR conditions in a mismatch repair assay wastested. This assay was developed specially for this purpose (U.S. Pat.No. 5,491,086) and comprises an amplification step and a diagnosticrestriction digestion. For the PCR reaction either primers (wild-typeprimers) are used which are totally homologous to the target DNAsequence (Taq DNA polymerase genes) and the 151 bp long PCR product ofwhich can be cleaved by the BamHI restriction enzyme, thereby generatinga DNA fragment 132 bp and 19 bp in size. Parallel to this, reactionswere carried out, the primer molecules of which have a base mismatch atthe 3′ end. If the incorrect base is not corrected during the PCR, theBamHI cutting site is destroyed and the PCR product cannot be cleaved ina restriction digestion with the enzyme BamHI. If on the other hand thepolymerase is capable of recognising wrongly paired bases andhydrolytically removing them, the correct complementary base can beincorporated and the PCR product yields the two expected DNA fragmentsafter restriction digestion. The wild-type primers used have thesequences: 5′-GCACCCCGCTTGGGCAGAG-3′ (SEQ ID NO: 6) and5′-TCCCGCCCCTCCTGGAAGAC-3′ (SEQ ID NO: 7). Alternatively, primers wereused the 3′ ends of which had a C:A, C:T or C:C mismatch. PCR reactionswere carried out with the Thermococcus pacificus DNA polymerase, Pfu DNApolymerase (Stratagene, Heidelberg, Germany), which has a mismatchcorrection, and Pfu Exo minus DNA polymerase (Stratagene, Heidelberg,Germany), which cannot perform this error correction. PCR reactionscontained 20 ng of plasmid pQE-31 (Qiagen GmbH, Hilden, Germany), whichcontained the Taq DNA polymerase gene target sequence, 1 unit of theappropriate DNA polymerase, 1× Pfu reaction buffer, 200 μM of each dNTPand 1.5 μM of each primer. The final volume of the reaction was 50 μl.The PCR reactions were carried out in an MJ Research PTC-200Thermocycler (Biozym, Hess. Oldendorf, Germany). The PCR programmecomprised an initial 1 min denaturing step at 94° C., a 30 secdenaturing step at 94° C., a hybridisation step at 62° C. and anextension step at 72° C. for 1 min. PCR products were purified with theQIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). Identicalamounts of PCR product were digested per 100 ng of PCR product with oneunit of BamHI for 90 min at 37° C. Then the PCR products thus treatedwere analysed on a 4% Metaphor Agarose gel (Biozym, Hess. Oldendorf,Germany). The results are shown in FIG. 3. The results show that, asexpected, the Pfu Exo minus DNA polymerase is unable to repair themismatch, i.e. the PCR product produced with mutated primers cannot becleaved by the restriction enzyme BamHI. By contrast the non-mutated PfuDNA polymerase is capable of correcting the base mismatch. TheThermococcus pacificus DNA polymerase is capable on the one hand ofsynthesising the desired PCR product under PCR conditions, and can alsorecognise mismatched bases under PCR conditions, remove themhydrolytically and replace them with the correct complementarynucleotide. Thus, it is clear that Thermococcus pacificus DNA polymeraseis suitable for carrying out so-called High Fidelity PCR reactions.

The invention claimed is:
 1. An isolated polypeptide having 5′-3′-DNApolymerase activity, which polypeptide is encoded by a DNA molecule,wherein said DNA molecule is the sequence of SEQ ID NO: 1, wherein saidpolypeptide further exhibits 3′-5′ exonuclease activity.
 2. A kit foruse in the polymerization of nucleic acid, said kit comprising: (a) apolypeptide according to claim 1; and (b) a reaction buffer for thepolymerization reaction; and optionally (c) dATP, dGTP, dCTP, and dTTP,either as a mixture or in individual containers.